QUANTITATIVE ANALYSIS OF CARBOHYDRATE-PROTEIN INTERACTIONS USING GLYCAN MICROARRAYS: DETERMINATION OF SURFACE AND SOLUTION DISSOCIATION CONSTANTS
A method, system and device to identify, study and/or mimic carbohydrate-protein interactions on cell surfaces and in solution measured by a glycan microarray. In some instances the method, system and device uses very small quantities of carbohydrate as low as attomol. In some instances the system, method and device is high-throughput. The small quantity senstivity may allow for close placement of carbohydrate array members wherein due to close proximity multivalent interactions with proteins may be identified.
This application is a divisional of U.S. patent application Ser. No. 12/422,733, filed Apr. 14, 2009, which claims the benefit and priority to U.S. Provisional Application Ser. No. 61/049,358, filed Apr. 30, 2008, the contents of each of which are herein incorporated by reference as if set forth in their entirety.FIELD OF DISCLOSURE
This disclosure related to sensitive, high throughput, glycan microarray systmes, methods and devices for examining carbohydrate-protein interactions on surface and in solution. More particularly, the glycan microarray is a platform for using very small amounts of materials. The glycan microarray supports mulivalent interactions and may be used to determine the surface dissociation constant (KD, surf).BACKGROUND
Carbohydrates can be envisioned as the K'nex™ toys of life. They are building blocks with multiple points of attachment, which can form highly brached and sterochemically-rich structures. They are difficult to study because the connecting points are not as sturdy as the K'nex. In fact, the binding force is very weak compared to the binding force of an antigen or antibody. The affinities, i.e., the force of attraction between molecules, of the latter can be 103-109 greater. Therefore, it is very difficult to synthesize a sufficient quantity of a carbohydrate for lab analysis. Traditionally it may take a day or more to measure a single carbohydrate-protein interaction using compounds in microgram to milligram amounts.
Carbohydrates, present as free oligosaccharides or as glycoconjugates, play an important role in many biological events, particularly those involving cell surfaces. Specific interactions between carbohydrates and proteins are often essential in viral and bacterial infection, the immune response, differentiation and development, and the progression of tumor cell metastasis. Therefore an understanding of carbohydrate-protein interactions at the molecular level would lead to a better insight into the biological process of living systems and assist the development of therapeutic and diagnostic strategies.
Despite the ubiquity and importance of carbohydrates in biology, difficulties in the study of carbohydrate-protein interactions have hindered the development of a mechanistic understading of carbohydrate structure and function. The structural compolexity of carbohydrates is a major obstacle: while the other two classes of biopolymers, nucleic acid and proteins, have a linear arrangement of repeating units, carbohydrate building blocks have multiple points of attachment, leading to highly branched and stereochemically-rich structrures. In addition, binding affinities are weak typicall in the ˜10−3-10−6 M range of dissociation constants, compared with antigen-antibody interactions (10−8-10−12). While techniques such as isothermal titration calorimetry (ITC), affinity capillary electrophoresis, surface plasmon resonance (SPR), and frontal affinity chromatography are all significant advances, they are often limited by the amount of available materials. Hence, the design of sensitive and high throughput technologies for characterizing carbohydrate-protein interactions remain a challenge.
However, little attention has been paid to the systematic kinetic and thermodynamic investigation of the interactions using glycan microarrays. Recently, MacBeath et al. reported a quantitative analysis of protein-peptide interactions using a protein microarray; in this work the interadctions of Src homology 2 and the phosphotyrosine binding domain of phosphopeptides were measured, and this study provided a better understanding of the tyrosine phosphorylation network for the epidemal growth factor receptor.
The glycan microarray or “sugar-chip” disclosed herein is a platform for the investigation and manipulation of carbohydrate-protein interactions. Assessing carbohydrate affinities is typically difficult due to weak affinities and limited sources of structurally complex glycans. Disclosed herein is a sensitive glycan microarry technology for the simultaneous determination of a wide variety of parameters in a single experiment using small amounts of materials. In some aspects this microarray is also high throughput.
In some exemplary implementations, a dense carbohydrate microarray for quantitative protein measurements is disclosed. The microarray may comprise: a solid substrate configured to provide a supporting substance; and a reactive layer fabricated on the solid substrate, the reactive layer comprising at least one reactive group of small molecules; wherein the at least one reactive group of small molecules is configured to bind with at least one protein, wherein the at least one protein are immobilized on the solid substrate via the reactive layer; wherein the carbohydrate microarray is configured to perform a quantitative measurement of binding ability with the at least one protein; wherein at least three different concentrations of the at least one protein are provided in one chip; wherein the binding affinity between the small molecules and the at least one protein can be measured; wherein the small molecules comprise at least one of carbohydrates and glycolipids; wherein the small molecules are disposed relative to each other to allow the at least one protein to multivalently bond with the small molecules.
In some exemplary implementations, a kit is disclosed. The kit may comprise: a high throughput carbohydrate microarray comprising carbohydrate spots on a solid substrate, each of the carbohydrate spots having a reactive group of at least one of carbohydrates and glycolipids, wherein the carbohydrates spots are configured to react with and bind to at least one protein; wherein the reactive groups are disposed relative to each other to allow the at least one protein to multivalently bond with the reactive groups; a microarray reader; and instructions for use.
In some exemplary implementations, a method for identifying a protein bound to a microarray of attomol quantity carbohydrates is disclosed. The method may comprise: forming a glycan microarray of spotted sugars in concentrations each of about 10−18 mole; adding a labeled protein to the microarray; incubating the micrarray; and, using an array reader to identify labeled proteins on the glycan microarray.
The above-mentioned features and objects of the present disclosure will become more apparatent with reference to the following description taken in conjunction with the accompanying drawings wherein like reference numerals denote like elements and in which:
In some exemplary implementations of the disclosure surface-based carbohydrate arrays is used to identify lectin recognition.
In some exemplary implementations of the disclosure the presentation of carbohydrates in a microarray provide a system and method to monitor multiple binding events and/or the effects of multivalency. In some instances implementations of the disclosure carbohydrate-proetein interactions on cell surface and in solution can be quantitatively measured by glycan microarray.
In some exemplary implementations of the disclosure a glycan microarray with attomol-limits of detection (1 attomol is 10−18 mol of a molecule) is diclosed. Thus, about one milligram of a carbohydrate may be used for a large number of tests. In some instances a milligram of carbohydrate may be used as many as 1012 times. In some instances very slight amounts of antibodies in a subject's blood stream, which can not be detected, by any other known method may be detected.
In some exemplary implementations a method for the determination of sugar density using fluorescein isothiocyanate cadaverine to discover, confirm and/or solve the distance of two binding sites within one protein.
In some exemplary implementations a method to mimic cell surface carbohydrate-protein interactions of at least one of binding mode and strength of carbohydrate-protein interaction.
In some exemplary implementations a glycans array to mimic cell surface carbohydrate-protein interactions of at least one of binding mode and strength of carbohydrate-protein interaction which uses a very small amount of carbohydrate.
In some exemplary implementations a method is disclosed to characterize sugar binding specificities of proteins.
In some exemplary implementations a method is disclosed for high thorughput identification of inhibitors of carbohydrate-binding proteins.
In some exemplary methods disclosed herein the apparent binding mode and strength of carbohydrate-protein interaction on cell surfaces are disclosed to be mimicked and can be quantitatively analyzed by glycan array in a rapid manner using only a very small amount of carbohydrate.
According to another aspect, one or more kits of parts can be envisioned by the person skilled in the art, the kits of parts to perform at least one of the methods herein disclosed, the kit of parts comprising one or more microarray devices, a solid substrate configured to provide a supporting substance; and a reactive layer fabricated on the solid substrate, the reactive layer compirising at least one reactive group of small molecules, according to implementations disclosed herein. The kits possibly include devices for reading, operating, interpreting, or processing data produced by the one or more microarrays, as well as instructions for use of the kit and its consituent parts. For example, a kit may include a microarray reader for analyzing a microarray after a reaction.
Arraying and detection limit. A strategy for covalently attaching a defined glycan to a glass slide was based on the standard microarray robotic printing technology using N-hydroxysuccinimide (NHS) activated glass surface, to which glycans containing an amine linked to the anomeric position were covalently attached. Prior to producing the slides, the challenges of printing concentrations were examined. Because carbohydrates do not fluoresce and modified carbohydrates bearing fluorescent groups might interact differently with the protein, fluorescein isothiocyanate (FITC) cadaverine was used as a model.
FITC cadaverine was printed in concentrations ranging from 100 mM to 1 fM and the slide was scanned before and after washing. The surface coverage of FITC was measured in a fluorescence wash-off experiment and the density of maximum loading was found to be 1014 molecules/cm2 a similar value to most peptides or sugars attached to SPR biosensor surface. More importantly, at concentrations below 100 μM, the fraction of surface covered by each molecule varied in proportion to its concentration, while over 500 μM, the surface seemed to be saturated (
Multivalent Carbohydrate-Protein Interaction on Surface. FITC labeled Con A was incubated with different printing concentrations of Man1 on the surface, after washing the slide was scanned to get the fluorescence intensities. A binding curve based on printing concentrations and fluorescence intensities was created, and the binding curve reached saturation (in the case of FITC-Con A and Man1, the curve became saturated at 10 μM printing conc.), which was independent of surface density (surface saturated when printing concentration was over 100 μM). This saturated curve is an indication of multivalent interaction between protein and printed carbohydrates. As the signal intensity in an array depends on the surfrace density of the immobilized carbohydrate, it is essential to normalize carbohydrate concentrations prior to printing.
In order to determine the dissociation constant on surface, protein concentrations were plotted against fluorescence intensity at different concentrations of printed sugar.
where Fmax is the maximum flourescence intensity, a measure of the amount of active carbohydrate on the surface; [P] is the total lectin concentration; and KD, surf the equilibrium dissociation constant for surface carbohydrate and lectin. Although the printed concentrations of mono-mannose vary by up to 10-fold from 100 to 10 μM, the KD, surf values obtained from these individual curves, as well as from replicate experiments, are narrowly distributed (mean KD, surface=83 nM; s.d.=4.7 nM;
The increase in binding strength for multivalent interactions is shown in the KD, surf values for high surface densities of a carbohydrate is the result of multivalent interactions. It is well known that carbohydrate-binding proteins interact weakly with monovalent ligands but strongly with multivalent carbohydrates. FITC washing-off experiment indicated that the average space between each sugar is about 100 Å at printing concentration of 1 μM. This result verifies FITC to be an appropriate model for the determination of sugar density1. Accordingly, in some exemplary implementations a method is disclosed to determine the distance of two binding sites within one protein. Applying at least some aspects of this method, different carbohydrates (Man1, Man4, Man8, Man9—see
The model dislcosed herein for binding fit the data well and KD, surf values were obtained using eq 1 (
We also compared the binding strength of lectins or antibodies to mannose derivatives at one or two concentrations of these proteins and obtained a ranking order for binding specificities. For example, the relative binding strength, based on maximal fluorescence intensitites, is Man4 (Fmax=36070)>Man9 (Fmax=25780)>Man8 (Fmax=13940)>Man1 (Fmax=9458), but that based on dissociation constants is Man8 (KD=320 nM)>Man9 (KD=335 nM)>Man4 (KD=490 nM)>Man1 (KD=3190 nM). Prior studies of carbohydrate-protein interactions have used a threshold-based, one-step qualitative analysis i.e. interaction or non-interaction. The threshold varies from one carbohydrate to another and is based on how well the carbohydrate behaves in the assay. Even when closely related glycans are studied under ideal conditions, they vary with respect to the surface density of active carbohydrates. Since the intensity of a spot depends both on KD, surf (which results from binding affinity), and Fmax (which results from surface active carbohydrate density and protein binding), the information obtained by probing an array with a single concentration of analytes may not be accurate. This present disclosures show that quantitative measurements can be carried out to accurately study the nature of carbohydrate-protein interaction on a cell surface.
Solution Dissociation Constant. The solution equilibrium dissociation constant (KD) for carbohydrate-lectin interactions can be determined using microarrays in a competitive assay. The analysis allows for the direct comparison between microarry affinities measurements to those obtained from solution-based affinity measurements. In a competitive binding experiment, carbohydrates in solution compete with immobilized carbohydrate ligands for the binding sites on the lectin, establishing a coupled equilibrium between the binding of proetin to the immobilized species and to the species present in the solution. Using array imaging signals, the unbound protein concentration [P] can be obtained via Langmuir isotherms (eq 1). Once the concentration of P has been measured, it is possible to determine the KD using eq 2, which is derived from the multivalent Scatchard formula (see the Experimental Section for the derivation of eq 2)
where [Lo] is ligand (carbohydrate) concentration applied to the system, and KD is the solution equilibrium dissociation constant. The derivation of this equation makes four assumptions: (1) the non-specific binding of protein to the slide surface is negligible compared to the total amount of protein in the system; (2) the binding sites in the protein bind to the ligand independently; (3) the initial concentration of ligand is much greater than the initial concentration of protein so that the concentration of unbound ligand is approximately equal to the total concentration of ligand (i.e., [Lo]≈[L]); and (4) the initial protein concentration for the system is greater than the initial concentration of protein-ligand complex (i.e., [Po]≈[P]). A competition binding experiment was performed by treating Con A to various oligomannoses, followed by incubation with corresponding oligomannoses surface. Binding curves, representing different concentrations of competitors were obtained as the function of FITC-Con A concentrations and fluorescence intensities from the Man8 surface (
Competitive Inhibitors of carbohydrate-Binding Proteins in Solution. When different inhibitors (such as α-methyl mannose (a-MeMan), α-methyl glucose (a-MeGlc), and etc.) are applied to the system, binding curves can be analyzed using eq 10 and the inhibition constant Ki can be obtained. Different concentrations of inhibitors were incubated with the slide bound with a protein of interest (
The glycan array system described here offers several features that make it attractive as a tool for glycomics: it requries small quantities of materials (10−18 mole) for high-throughput analysis, and can be used for quantitative analysis of carbohydrate-protein interaction on surface and in solution. The disclosed system is considered to be a good mimic of cell-surface arrays of carbohydrates in which the dissocation constants of multivalent interactionscan be determined for comparison with the monovalent, solution dissociation constants determined through the competition analysis.EXAMPLE 1
Materials. NHS-coated glass slides (Nesterion H slide, SCHOTT North America; high density amine binding slide, Amersham bioscience), FITC labeled Concanavalin A (Con A, Sigma), FITC labeled Lens culinaris agglutinin (LCA, Sigma) and FITC labeled Pisum sativum agglutinin (PSA, Sigma), α-methyl mannose (Vecotr laboratory), α-methyl galactose (Sigma). Mannose derivatives (Man1, Man4, Man8, Man9) were synthesized as previously described.EXAMPLE 2
Microarray Fabrication. Microarrays were printed (Genomic Solutions, Gene Machine) by robotic pin (SMP2B, TeleChem International Inc.) deposition of ˜0.6 nL of various concentrations of amine-containing glycans in print buffer (300 mM phosphate, pH 8.5 containing 0.005% Tween-20) from a 384 well plate onto slides. The slide for (1) the scope of printing concentration studies: NHS-coated glass slides were printed with Man1 and FITC at 30 concentrations between 0.5 fM to 100 mM from left to right with 16 replicates vertically placed in each sub-array consisted of a 30×16 pattern of spots, with a 0.25 mm pitch. After 1 h reaction, the slides surface was divided by drawing with permanent marker to avoid contamination for later protein incubation; (2) the slide for
Fluorescence wash-off measurements. FITC cadaverine (
Since the printing concentration, C=500 μM or 100 μM, and the volume, V=0.6 nL, NA is Avogadro's Number. Each spot in the array is around 0.1 mm diameter.
When the surface is saturated (around 100 mM to 500 mM printing FITC), the ratio from pre-quench (Qpre) to post-quench (Qpost) is 0.23 (obtained from the array scanner measurement before and after washing). Based on equation 3, the surface density is between 1.8×1014 molecules/cm2 and 8.8×1014 molecules/cm2. When the printing concentration at 1 mM, the ratio of Qpost/Qpre is 0.1, the density is 0.77×1012 molecules/cm2. If the surface was assumed to be of a homogenous distribution, the average space between each molecule is 115×10−8 m (115 Å).EXAMPLE 4
Direct Binding Assay. FITC-labeled Con A (4 mg/ml), FITC-labeled PSA (2 mg/ml), FITC-labeled LCA (2 mg/ml), were diluted in PBS-BSA buffer 50 mM, pH 6.5; 1 mM CaCl2, 1 mM MnCl, 0.9% NaCl (w/v), 1% BSA (w/v)). Human monoclonal antibody-2G12 was used in PBST buffer with 1% BSA. For all incubations, 10-25 μL of protein solution was applied to each sub-array using a 24 well bottomless incubation chamber (The Gel Company). Humidifying incubation was performed under foil and using a shaker for 1 h at room temperature. The slide was washed three times with incubation buffer, three times with PBST buffer (0.05% Tween-20), three times with distilled water, and then centrifuged at 200 g for 5 min to ensure complete dryness. The array was then imaged at a resolution of 5 Å mwith a A488 laser using ArrayWorx microarray reader to visualize fluorescence. The method for the interaction of human monoclonal antibody-2G12 with sugars was similar to lectin. 2G12 in PBST buffer with 1% BSA was pre-complex with Cy3 labeled goat anti-human IgG (Jackson ImmunoResearch) and then placed to the slide and incubated for 1 h. The images were read using A595 laser with the array reader.EXAMPLE 5
Competitive Binding Assay. 15 μl of series dilutions of competitor was incubated with different concentrations of protein (15 μl). The mixture was then loaded onto the slides by using a 24 well incubation chamber and incubated for 1 h under a humidifying container at room temperature. The following procedure is the same as the direct binding assay.EXAMPLE 6
Data analysis. ArrayVision 8.0 (Applied Precision) was used for the fluorescence analysis and extraction of data. Equilibrium binding data were analyzed by fitting the data to the appropriate equation, assuming that ligands bound to one or two independent sites, using the commercial non-linear regression program GrapPad PRISM (GraphPad). The error bars indicated in the figures show the average percentage error for all data points reported in the figures.EXAMPLE 7
Calculation. The formation of surface-bound complex (LP) on the slide between analyte protein (P) and surface bound ligand (L) can be generally considered to the simple bimolecular reversible reaction scheme.
The observed rate of complex formation may be written
The concentration of unoccupied ligand [L] is the difference between the total amount of ligand [Lo] and the amount of [LP]. Substituting [Lo]-[LP] for [L] in eq 4 gives
if the total amount of ligand [Lo] is expressed in terms of maximum analyte binding capcity of the surface, all concentration terms can then be expressed a binding signal response (F).
When at equilibrium d[F]/dt=0 and KD, surf=kd/ka, the dissociation constant of ligand and protein complex is obtained as shown in eq 1.
The interaction between a monovalent ligand (L) in the solution, a monovalent ligand (L) on the surface, and a multivalent protein (P) can be represented as
The expression for the equilibrium solution dissociation constant for this interaction is
where [LP*] is the concentration of protein/ligand complexes, [P] is the concentration of free protein, and [L] is the concentration of free ligand. Since a multivalent protein (P) may have q binding sites (B), the concentration of free binding sites [B] is equal to q[P]. Likewise, the formation of a binding site/ligand complex [BP*] is equal to q[LP*]. From the eq 7, both numberator and denominator muliply q value. The interaction of one acceptor binding site with ligand can be represented as
Since the binding sites in the protein bind to the ligands independently, the individual dissociation constant is therefore the same as protein dissociation constant.
Then, a value is defined as ratio of free protein and total protein which is then substituted [LP*] value by eq 7 and rearranged to give eq 9.
To determine KD, the unbound protein in this system is calculated to become a[Po] and which is substituted to eq 1 and rearranges to yield eq 2.
The interaction between inhibitors (I) in the solution, a ligand (L) on the surface and multivalent protein (P) can be represented as
The array imaging data is used to measure [P] and Ki can be determined by the eq 10.
The fraction of inhibition (f) is equal to 1-F/Fmax, the equation from eq 10 can be rearranged to give eq 11.
While the method and agent have been described in terms of what are presently considered to be the most practical and preferred implementations, it is to be understood that the disclosure need not be limited to the disclosed exemplary implementations. It is intended to cover various modifications and similar arrangements included within the spirit and scope of the claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures. The present disclosure includes any and all implementations of the following claims.
1. A carbohydrate microarray for protein measurements, comprising:
- a solid substrate configured to provide a supporting substance; and
- a reactive layer fabricated on the solid substrate, the reactive layer comprising at least one group of small molecules wherein the small molecules comprise at least one of carbohydrates and glycolipids wherein the small molecules are disposed relative to each other to allow the at least one protein to multivalently bond with the small molecules;
- wherein the at least one group of small molecules is configured to bind with at least one protein wherein the at least one protein is immobilized on the solid substrate via the reactive layer;
- wherein the carbohydrate microarray is configured to perform quantitative measurement of binding with the at least one protein; and
- wherein at least one protein of a given concentration is applied whereby the binding affinity between the small molecules and the at least one protein can be measured.
2. The device of claim 1, wherein an average spatial separation between the small molecules ranges from about 65 Angstrom to about 100 Angstrom.
3. The device of claim 1, wherein the proteins comprise at least one of carbohydrate binding proteins and lipid binding proteins.
4. The device of claim 1, wherein the proteins are fluorescence labeled or can be detected by a secondary labeled protein.
5. The device of claim 1, wherein each of the at least one reactive group has at least about 10−18 mole of carbohydrate.
6. The device of claim 1, wherein carbohydrates with reactive groups are immobilized on the surface of the device wherein the arrays comprise about 2 to 50 subarrays wherein each subarray contains one or more carbohydrates.
7. A kit comprising:
- a carbohydrate microarray comprising carbohydrate deposited on a solid substrate, each carbohydrate comprising a reactive group of at least one of carbohydrate and glycolipid, wherein the carbohydrates are configured to react with and bind to at least one protein; wherein the reactive groups are disposed relative to each other to allow the at least one protein to multivalently bond with the reactive groups;
- a microarray reader; and
- instructions for use.
8. The kit of claim 7, wherein the carbohydrates of the microarray are deposited at least about 10−18 mole of carbohydrate molecule per spot.
9. The kit of claim 7, wherein the carbohydrates include at least one glycan.
10. The kit of claim 7, wherein the carbohydrates include at least one glycolipid.
International Classification: G01N 33/53 (20060101);